Baculoviruses are large DNA viruses that are virulent pathogens of insects and they serve as important models for studies of virus-host interactions. In addition, the baculovirus AcMNPV has been developed as a transduction vector for mammalian cell expression, with important applications in areas such as high throughput screening and the potential for use in human gene therapy. Viral entry by AcMNPV budded virions (BV) is mediated by the major envelope glycoprotein, GP64. The receptor for GP64 is not known. GP64 is also necessary for efficient virion budding and progeny virus production. In natural hosts of AcMNPV, the GP64 protein also plays a critical role in the initial phase of transmission in the insect. GP64 is targeted to basal membranes in polarized midgut epithelial cells and this targeting appears to direct budding and transmission of infection into the insect hemocoel. In the proposed studies, our work will be concentrated in three specific areas: 1) Viral receptor binding;2) Envelope protein targeting in insect polarized midgut epithelial cells;and 3) Virion assembly and budding. Studies of viral receptor binding will focus on identification of the GP64 receptor binding domain and the host cell receptor. We will also identify the GP64 midgut targeting signal and interacting proteins involved in this process. Because GP64 is critical for efficient virion budding, we will identify the GP64 budding domain as well as viral and/or cellular proteins that interact with GP64 during assembly and budding. For these studies, we will use an engineered cell line expressing GP64, and a powerful genetic system that we recently developed, to replace wild type gp64 in the viral genome with modified forms of gp64. Using these powerful genetic tools with a range of functional assays, we will examine GP64 function in the context of budded virions and the viral infection cycle. These studies will address important and central questions, advancing our understanding of baculovirus interactions with host receptors, virus movement through insect polarized midgut epithelial cells, and the mechanism of virus budding. In addition, results of many of these studies will be directly applicable to new and exciting biotechnological applications of baculoviruses in mammalian cells.